Illnesses caused by rickettsiales of the genera Ehrlichia and Anaplasma are a growing human health concern in recent years and are the second leading cause of tick-borne infections in the USA and many parts of the world. They include human monocytic ehrlichiosis caused by Ehrlichia chaffeensis. Despite the sophisticated systems of defense in vertebrate and tick hosts, the rickettsiales evade the host clearance. E. chaffeensis in macrophages and tick cells differ significantly in expression of several outer membrane proteins and effector proteins secreted via its secretary pathways. Research results demonstrate that differential tick and vertebrate host cell-specific expression contributes to the varied host response and delayed clearance in a host. The central hypothesis of the previously funded project is that E. chaffeensis differentially regulates gene expression and that the host-specific gene expression is essential for its survival in vertebrate and tick cells. Progress from the previous funding cycle forms the strong foundation for this renewal application and to continue testing the above stated innovative hypothesis. Specific aims of this application are 1) to characterize E. chaffeensis RNA polymerase complex in support of understanding how vector and vertebrate host-specific differential gene expression is accomplished, 2) to evaluate the significance of host-specific differential expression by characterizing mutations in three genes identified as essential for E. chaffeensis in vivo growth, and 3) to perform mutational analysis and in vivo screening to identify genes essential for the E. chaffeensis pathogenesis.